The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation

HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5′ and 3′ end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal

HIV Tat and Morphine-induced Neurodegeneration in a Beclin 1 Hemizygous Mouse Model

Early in infection, HIV crosses the blood-brain barrier and induces neuropathology. Viral presence in the CNS coupled with secretion of neurotoxic proteins causes neuroinflammation, glial dysfunction, excitotoxicity, and neuronal death. Despite advances in combined antiretroviral therapy, HIV-infected patients present with a spectrum of cognitive and psychomotor deficits collectively referred to as HIV-associated neurological disorders (HAND). A subset of HAND patients abuses drugs such as opiates like heroin and morphine show an exacerbation and rapid progression of HIV neuropathology; however, the mechanisms of this synergy are not well understood. Autophagy is a lysosomal degradative process which eliminates and recycles cytosolic components and is implicated in facilitating HIV-1 replication in the CNS and periphery, and in Tat-induced neurodegeneration. When a key initiator of autophagy Beclin 1 was silenced using siRNAs, there was a marked reduction of HIV-1 replication in human microglia and astrocytes and the corresponding inflammatory response. As such, the goal of the current study is to determine if diminished Beclin 1 is neuroprotective against Tat and morphine-induced neurodegeneration using heterozygous Beclin 1 (Becn1+/-) mice. Examination of Tat and morphine-induced inflammatory molecule secretion revealed that Becn1+/- mixed astrocyte and microglia (glia) exhibited attenuated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to control (C57BL/6J) glia, an effect mediated through the μ-opioid receptor. Dysregulation of autophagy-related gene expression and excessive intracellular calcium accumulation were limited in Becn1+/- glia. When determining the effects of Tat-and morphine co-exposure on neuronal survival in vitro, we found Becn1+/- neurons were particularly sensitive to injury, excitotoxicity, and toxic exposures; however, when C57BL/6J neurons were exposed to conditioned media of C57BL/6J and Becn1+/- glia treated with Tat and morphine, neurons treated with Becn1+/- supernatant had better outcomes than those treated with C57BL/6J conditioned media. Furthermore, despite minimal difference between strains in locomotor assessment, we observed significantly greater striatal neuron losses in adult C57BL/6J mice exposed to intrastriatal Tat-and systemic morphine compared to Becn1+/- mice. Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction

Engineering Signal Transduction Pathways in Bacteria

Veränderte Proteine sind ein wertvolles Handwerkzeug, sowohl in der Grundlagenforschung als auch in der Industrie. Ein nächster Schritt wäre Veränderungen in biologischen Stoffwechselwegen, z.B. in der Signalweiterleitung, vorzunehmen. Dieses könnte die Grundlage für viele Anwendungen sein, z.B. in der Gentherapie um neue Eigenschaften in Organismen einzuführen. Viele biologische Prozesse sind einem hohen Rauschpegel unterworfen, was für viele Anwendungen nicht geeignet ist. So können z.B in einer Population von Zellen die Gene unterschiedlich stark aktiviert werden, was zu niedriger oder hoher Expression führen kann. Daher ist es wichtig beim Entwurf von neuen Signalwegen, dass der Output gut kontrolliert werden kann. Da man solch einer Herausforderung nicht einfach gerecht werden kann, ist es besser mit einfachen und gut analysierten Signalwegen, wie sie z.B. in E.coli vorkommen, anzufangen. Die Signalweiterleitung in E.coli erfolgt hauptsächlich in Zwei-Komponenten Systemen. Diese bestehen aus einer Kinase als Sensor und einem Regulatorprotein, welches normalerweise ein Transkriptionsfaktor ist, mit einigen Ausnahmen, wie z.B CheY aus dem Chemotaxis Signalweg. In dieser Arbeit wurden zwei gut charakterisierte Signalwege durch einen chimärischen Sensor verbunden, um so einen neuen Signalweg herzustellen: Die Ligandenbindungsdomäne des Aspartat Receptors, Tar, aus dem chemotaxischen Signalweg wurde mit der katalytischen Domäne des osmosensorischen EnvZ kombiniert, um den chimärischen Rezeptor Taz herzustellen. Taz kann die Genexpresion aktivieren durch Phosphorylierung des Antwort-Regulators OmpR, über den Porin Promotor, nach Bindung eines geeigneten Liganden, wie z.B. Aspartat. Ziel dieser Studie war es dieses System zu nutzen um einen Signalweg herzustellen, dessen Output durch einen Gen-Schaltkreis kontrolliert ist, und das Eingangssignal durch gezieltes Design zu ändern. Als Output wurde ein etabliertes GFP verwendet und unterschiedliche Schaltkreise wurden verwendet: a) Kompetition von OmpR-P mit dem TetR Repressor, expremiert von einem synthetischen Promotor, zur Aktivierung des pompC Promotors, b) Expression von Antisense RNA für das Reportergen (GFP) and c) einen bistabilen Schalter durch TetR und einem temperatur-abhängigen Protein CI, welches durch OmpR-P aktiviert wird und die größte Regulation des Outputs ist. Trotz dieser unterschiedlichen Ansätze war es nicht möglich ein stabiles Konstrukt zu erhalten mit ausreichender Promotor Stärke, um einen Effekt aufzuweisen im Vergleich zu dem einfachen Reporter pompC-GPP. Das Liganden-Design erfolgte mit Hilfe der Algorithmen PERLA und FoldX sowie dem Programm SwissPdbViewer, basierend auf der Kristallstruktur der periplasmatischen Domäne von Tar im Komplex mit Glutamat. Obwohl diese Methode sehr vielversprechend aussah, als sie für einige Mutaten in dem Wildtyp Chemotaxis System durchgeführt wurde, war sie nicht erfolgreich um die Liganden des Chimären Rezeptors zu ändern. Ein Grund hierfür könnte sein, dass der chimäre Rezeptor in der periplasmatischen Domäne eine andere Konformation als der Wildtype Rezeptor hat, als Folge der Fusion mit der cytoplasmatischen Domäne von EnvZ. Dies wird durch experimenteller Ergebnisse unterstützt, die zeigen, dass Tar und Taz nicht dieselben Erkennungs-Eigenschaften haben, so z.B. können gleiche Signale für Tar entgegengesetzte Antworten durch Taz hervorrufen. Dennoch, in dieser Studie wurden in großem Detail die Eigenschaften von Taz untersucht, was zur ersten Entdeckung von Liganden (Aminosäuren) führte, die den Rezeptor inhibieren können. Weiterhin konnte gezeigt werden, dass diese Inhibierung stereo-spezifisch ist und extrem stark ist, sogar stärker als dieBindung von Liganden wie Aspartat. Weiterhin ergaben die intrinsischen Eigenschaften des Taz-OmpR Systems, die in dieser Studie gefunden wurden, eine neue Ansicht für das Wildtyp-System: Die Aktivierung des EnvZ-OmpR Systems in E. coli während des Zellwachstums scheint über EnvZ zu erfolgen, und die katalytische Domäne von EnvZ ist nicht ausreichend um dafür. Der wichtigste Befund aber für das chemotaxische System ist, dass einige der inhibitorischen Aminosäuren für Taz zu einer neuen, komplizierten Antwort führten und somit die Existenz eines bisher uncharakterisierten Adaptation Signalweges aufgedeckt wude

Influence de l'initiation de la traduction sur le changement programmé du cadre de lecture en -1 responsable de la synthèse des enzymes du virus de l’immunodéficience humaine de type 1

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage.The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift

G-QUADRUPLEXES IN THE HIV-1 GENOME AS ANTIVIRAL TARGETS

G-quadruplexes are non-canonical nucleic acid structures: their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they act as silencers in the promoter regions of human genes and putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes. Moreover, they have been proposed to be directly involved in gene regulation at the level of transcription. We investigated G-quadruplex formation in the HIV-1 proviral genome to assess the potential for viral inhibition through G-quadruplex stabilization. Here we show that the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity. In addition, we found three conserved putative G-quadruplex forming sequences uniquely clustered in the coding region for the accessory protein Nef that were efficiently stabilized or induced by G-quadruplex ligands. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. Finally, a comprehensive screening of G-quadruplex ligands against HIV-1 disclosed significant potential of some of them as HIV-1 inhibitors, likely with a G-quadruplex-mediated mechanism of action. These findings open up the possibility of inhibiting the HIV-1 by G-quadruplex-interacting small molecules, providing a new pathway to the development of anti-HIV-1 drugs with unprecedented mechanism of action

Crosstalk of lysosomes, autophagy and apoptosis in dioxin-induced chloracne in vitro

PhD ThesisChloracne is a hyperkeratotic acneform skin disease caused by exposure to 2,3,7,8-tetrachlorodibenzo–p-dioxin (TCDD), a potent agonist of the aryl-hydrocarbon receptor (AhR). Despite the known role of TCDD exposure in the pathogenesis of chloracne, the molecular mechanisms mediating the disease remain poorly defined. Using a previously optimised in vitro primary human keratinocyte epidermal equivalent model, we demonstrated the temporal regulation and development of a significantly reduced viable cell layer and compacted stratum corneum over 7 days with 10nM TCDD. These morphological changes were paralleled by cumulative AhR protein degradation and increased mRNA levels of CYP1A1. One of the key findings was a significant increase in the expression of the apoptotic marker, caspase-3 whilst there was no significant effect on Ki67 staining. Furthermore, TCDD treatment caused de-regulated epidermal differentiation as evidenced by increased mRNA expression but decreased protein expression of late differentiation markers. Treatment with TCDD also resulted in an induction of LC3 lipidation and endogenous LC3 protein expression as well as decreased P62 protein expression, well-established markers of autophagy induction. Interestingly, co-treatment of epidermal equivalents with TCDD and the lysosomal inhibitor, bafilomycin or cathepsin D inhibition (by pepstatin A or shRNA knockdown) resulted in restoration of the viable cell layer and reduction in TCDD-induced caspase-3 expression. Similar results were also seen after blockade of the autophagy pathway by ATG7 knockdown. Results also demonstrated lysosomal function and autophagy are required for TCDD-induced AhR degradation, indicating a potential role for chaperone-mediated autophagy. Collectively these data suggest exposure to TCDD results in deregulated epidermal differentiation, induction of autophagy, reduction of a viable cell layer and caspase-3 dependent cell death, likely mediated via lysosomal processing. Results provide an insight into the pathophysiology of chloracne, and demonstrate novel findings including TCDD-induced autophagy and potential role of lysosomal function in TCDD-induced death and AhR degradation.Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca

Synthesis of different series of small molecules targeting HIV-1 RT, Candida albicans, MAO and G-Quadruplex

My PhD work has been focused towards four different targets: HIV-1 RT, Candida albicans, Monoamine oxidase, and G-Quadruplex. Thus in order to give the reader a clearer exposition this report has been divided in four different chapters. Each of the chapters has his own figures, schemes, tables and references. The main part of my work has been dedicated to HIV-1 RT, thus this chapter is the major and first one